Despite the advent of the Papanicolaou (Pap) smear, cervical cancers and pre-cancers remain important health problems for women, especially underobserved women in the United States (U.S.) and women in underdeveloped countries (Parkin, 1993). The incidence of both invasive cervical cancer and carcinoma-in-situ are increasing in the U.S. The reasons for this increase are unknown. In the U.S., an estimated 2,500,000 women will have abnormal Pap smears demonstrating atypical cells of uncertain significance and low-grade intraepithelial lesions (lesions of HPV and CIN 1) annually (Kurman, 1994). The exact number of patients with high-grade squamous intraepithelial lesions (CIN II and III), not classified as carcinoma-in-situ, is unknown.
A risk factor for cervical cancer is HPV. The most common types of HPV are those classified as high risk (HPV 16, 18, 45, and 56), intermediate risk (HPV 31, 33, 35, 51, 52, and 58), and low risk (HPV 6, 11). The high and intermediate risk types have been identified in 77% of high grade cervical intraepithelial neoplasia (CIN) and squamous intraepithelial lesions (SIL) and in 84% of invasive lesions. Cohort studies demonstrate that women with HPV infection have 11-60 times increased risk of developing high grade CIN and 15-50 times increased risk of developing invasive cancer than do women without HPV infection for which the high-risk HPV types include types 16, 18, 45, and 56. This association has been consistent and independent of the HPV assay method employed or epidemiologic study design (Bosch, 1995).
Chemoprevention is the use of chemical agents (micro-nutrients, pharmaceuticals) to prevent or delay the development of cancer in healthy populations (Kelloff, 1994; Daly, 1993). These agents, which block the initiating and promoting events of carcinogenesis, augment the preventive strategy, which includes the avoidance of carcinogens in the environment (referred to as primary prevention) and participation in screening programs (referred to as secondary prevention), hence serving as a tertiary preventative measure.
Because most current chemopreventive agents can cause side effects, they are used in individuals who have higher risk of developing cancer (for example, those with premalignant lesions). Intervention in the precancerous stage may prevent a lesion from becoming invasive (Spom, 1993). The advantage of chemoprevention in the treatment of a pre-neoplastic condition is that the effects of chemoprevention are systemic, thus pre-neoplastic cells in all areas of the body are treated. Chemoprevention studies use surrogate endpoint biomarkers (SEB's) as intermediate measures of cancer development. These markers should be differentially expressed in normal and high-risk tissue, be measured with acceptable sensitivity and specificity, and be modulated by the chemopreventive under study. SEB's provide a glimpse of cancer biology and its modulation (Boone, 1990).
There are several reasons why chemoprevention is attractive for cervical lesions. These reasons reflect the belief that pre-cancers, like cancers, represent a systemic process. Many colposcopy patients smoke, and many of these patients also have preneoplastic and neoplastic lesions of the aerodigestive tract. Infection with HPV affects the entire squamous epithelium of the female genital tract, and up to 40 % of patients with CIN have multifocal lesions of the vagina, vulva, and perianal area.
Polyamines (putrescine, spermidine, and spermine) and their precursors (arginine and ornithine) are thought to play critical roles in cellular maintenance, proliferation, differentiation, and transformation; thus polyamines might be considered surrogate endpoint biomarkers of carcinogenesis. Polyamines are differentially expressed in normal and high-risk tissue; measured with acceptable sensitivity and specificity; and can be modulated by DFMO. Polyamines and their precursors can be measured in tissue, red blood cells, plasma, and urine. Ornithine Decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is considered a proto-oncogene that is crucial for the regulation of cellular growth and transformation and is irreversibly inhibited by the drug .alpha.-difluoromethylornithine (DFMO). DFMO is considered a potent anti-proliferative chemopreventive agent and has been studied in other organ sites but has not previously been studied in the cervix (Verma, 1987; Auvinen, 1992).
ODC catalyzes the first step in the biosynthesis of putrescine (a diamine), spermidine and spermine, the three major polyamines of mammalian cells. In vitro studies show that polyamines participate in nearly all aspects of DNA, RNA, and protein synthesis. Polyamine accumulation is required to maintain maximum rates of cell proliferation. Blockage of polyamine accumulation by administration of DFMO and other inhibitors during accelerated cell growth results in a significant reduction of growth in a variety of cell systems. Since the only pathway to polyamine synthesis is via ornithine, synthesis depends on the activity of ODC. ODC is present in very small amounts in resting cells but can be increased many-fold within a few hours of exposure to hormones, drugs, and growth factors.
Blocking endogenous ODC has reportedly prevented transformation of rat fibroblasts by the temperature-sensitive v-src oncogene. Tumor formation in experimental animals has reportedly been prevented by inhibitors of ODC such as DFMO. DFMO has reportedly been shown to inhibit cellular replication in vitro in several malignant animal tumor cell lines, including L1210 and L5178Y leukemia, rat hepatoma, mouse mammary sarcoma (EMT6) and hamster pancreatic adenocarcinoma (H2T) (Mamont et al., 1978; Prakash et al., 1980; Marx et al., 1987).
DFMO, either alone or in combination with other agents, has proven effective in treating and/or preventing mammary carcinomas in animal models as follows. The growth of six human tumors (three mammary carcinomas, a malignant melanoma, a bladder carcinoma, and an endocervical carcinoma) was significantly decreased after DFMO treatment compared to growth in control mice. (Luk et al., 1983). In xenographs of human breast and colon carcinoma cells inoculated into nude mice, a synergistic antitumor effect was observed when DFMO was combined with mitomycin D (Takami et al., 1989).
Studies by Nishioka et al. report increases of plasma precursor amino acids of polyamines, such as arginine and ornithine, at low doses of DFMO in tissue obtained by routine cervical biopsy. DFMO was also measured as a compliance marker. Nishioka et al. proposed that polyamines and their precursor amino acids would be effective markers in analyzing the effects of DFMO, functioning as pharmacodynamic parameters, as well as biomarkers for transformation in the cervix.
Chemoprevention trials of cervical lesions using topical retinoic compounds and micronutrient have been reported; most of them being considered as negative trials. A trial by Meyskens reports histological regression of CIN II cases, but not CIN III, using topical trans-retinoic acid. The rate of regression in the treated CIN II group was reported to be 43 %, compared to 27% in the placebo group.
Determining the optimal dose of DFMO could be based on several parameters, including toxicity, modulation of polyamine synthesis markers, and/or response. In the Phase I study of Love, a dose of 0.5 g/m.sup.2 /day was used, being based on toxicity (Love, 1993). Studies employing modulation of polyamine synthesis markers to determine optional dose of DFMO, specifically urine polyamine markers putrescine and spermidine at a DFMO dose level of 0.2 g/m.sup.2 /day, have been reported in Meyskens, 1994 and Hixson, 1993. Employing polyamine synthesis markers, Meyskens, (1994) reports modulation of tissue spermidine/spermine ratio at a dose of 0.1 g/m.sup.2 /day. A need continues to exist in the medical arts for methods of detecting, inhibiting and reducing the risk of developing pre-malignant tissues linked to cervical malignancies.